E16 c57bl 6 mice

Cortical neuron primary cultures were prepared from E16 C57BL/6 mice (Charles River). The pregnant mouse was anesthetized using isoflurane (5%), and embryos were transferred to ice-cold HEPES-Glucose buffer (HEPES 10 mM (Fisher), Glucose 33 mM (Fisher) in PBS (Corning)). Embryos were decapitated and the cortices were dissected from the rest of the brain and incubated in HEPES-Glucose buffer containing Trypsin, at 37 °C, for 15 minutes. Trypsin was washed by HEPES-Glucose buffer, and replaced by DMEM (Dulbecco's Modified Eagle Medium, GIBCO) fortified with 10% FBS (Fetal Bovine Serum, GIBCO). Cortical tissues were fragmented by forceful pipetting using glass Pasteur pipettes, and then plated in 24-well culture plates at a density of 20,000 per cm². Cultures were incubated at 37 °C for 2 hours. After this time, the culture media was replaced with Neurobasal medium (GIBCO) fortified with B27 supplement (2%, GIBCO) and Glutamax (0.25 %, GIBCO). After 5 days in vitro, the cultures were transfected with adeno-associated viruses carrying shRNA against the target protein’s mRNA. Cultures were then incubated for 10 days at 37 °C. Neuronal lysates were subsequently collected using lysis buffer (SDS 2%, Tris 50 mM, EDTA 2 mM).

Brain cortices were collected from embryonic day 16 (E16) C57/Bl6 mice (Charles River Laboratories), following the established protocol.^{[57 ]} In brief, mouse cortices were freshly dissected and stored in HBSS solution for the duration of the dissection. Prior enzymatic digestion, cortices were washed twice with cold PBS. A cortical cell suspension was prepared by enzymatic digestion with a 2mL mixture of trypsin/EDTA (0.25%) (ThermoFisher) combined with DNase (final concentration 0.3mg/mL; Roche Applied Sciences). After 20min incubation at 37°C, Trypsin activity was inhibited by adding an equal volume of Neural Medium (NeuroBasal medium supplemented with 2% B27, 100U/mL penicillin, 100 ug/mL streptomycin, 2mM Glutamax (ThermoFisher)) and 10% of heat inactivated fetal bovine serum (hiFBS) (ThermoFisher)). The cell suspension was passed through a 100μm filter to remove cell aggregates and counted. Cells were then concentrated by centrifugation and resuspended in fresh neural medium at a cell density of 25 million cells per ml of Neural Medium. All animal procedures were approved by the Tufts University Institutional Animal Care and Use Committee and complies with the National Institutes of Health Guide for the Care and Use of Laboratory Animals (Institutional Animal Care and Use Committee M2018–06).